CN114106154B - Chromatography method of nano antibody - Google Patents
Chromatography method of nano antibody Download PDFInfo
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- CN114106154B CN114106154B CN202210082970.5A CN202210082970A CN114106154B CN 114106154 B CN114106154 B CN 114106154B CN 202210082970 A CN202210082970 A CN 202210082970A CN 114106154 B CN114106154 B CN 114106154B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
Abstract
The invention relates to a chromatography method of a nano antibody, which comprises the following steps: s1: carrying out affinity chromatography on the nano antibody harvest solution, and collecting an affinity chromatography eluent; s2: adjusting the pH of the affinity chromatography eluent to 5 +/-0.5, and the concentration of the nano antibody to be less than or equal to 6mg/mL, and then adding (NH)4)2SO4Adjusting the conductivity to 80 +/-20 mS/cm, and preparing a to-be-chromatographed liquid; s3: and carrying out hydrophobic chromatography on the liquid to be chromatographed. The chromatography method of the nanobody effectively removes aggregates, Host Cell Proteins (HCPs) and host cell dna (hcd).
Description
Technical Field
The invention relates to the technical field of antibody purification, in particular to a chromatography method of a nano antibody.
Background
Antibodies (antibodies) are globular proteins produced by the body's immune system against foreign substances (e.g. proteins of bacteria, viruses) and are therefore also referred to as immunoglobulins (Ig). In recent years, as antibody drugs are increasingly applied to more therapeutic fields, the structures of antibody drugs have become more diversified. Among them, the nano antibodies (Nbs) have unique advantages compared with the conventional monoclonal antibodies, including relatively weak immunogenicity, enhanced tissue permeability and the like, due to their small relative molecular mass, and their unique molecular structures also make them suitable for many fields such as disease diagnosis and treatment.
Antibodies are generally purified by chromatography for use. Chromatography methods against traditional monoclonal antibodies may be as follows:
methods exist for purifying antibody molecules using hydrophobic interaction chromatography in combination with chromatography, which may include the sequential steps of protein A affinity chromatography, ion exchange chromatography, and hydrophobic interaction chromatography. The method enables removal and recovery of monomeric IgG antibodies in the antibody free of immunoglobulin aggregation, misfolded species, and Host Cell Proteins (HCPs) and protein A.
Still another method provides a method for purifying a human glucagon-like peptide-1 (hGLP-1) analog fusion protein, including dolabrin, comprising purifying by three-step chromatography: the sample is subjected to Protein A affinity chromatography of a coarse purification step to effectively remove impurities such as HCP, endotoxin and the like and maintain higher yield of target Protein; then further fine purification is carried out by using anion exchange chromatography and hydrophobic chromatography to effectively remove charge isomers, residual HCP and other trace impurities in the sample, control the content of each impurity within a drug safety range and maintain higher yield and activity of the target protein.
However, the molecular design of the existing nanobody is not fully mature, and compared with the traditional monoclonal antibody, the nanobody has the problems of poor stability and easy aggregation, and the generated aggregates and impurities such as HCP, Host Cell DNA (HCD) and the like are more difficult to remove by the traditional chromatography method.
Disclosure of Invention
Based on this, the present invention provides a chromatography method of nanobody capable of effectively removing aggregates, Host Cell Protein (HCP) and host cell dna (hcd).
The specific technical scheme is as follows:
the invention provides a chromatography method of a nano antibody, which comprises the following steps:
s1: carrying out affinity chromatography on the nano antibody harvest solution, and collecting an affinity chromatography eluent;
s2: adjusting the pH of the affinity chromatography eluent to 5 +/-0.5 and the concentration of the nano antibody to be less than or equal to 6mg/mL, and then adding (NH)4)2SO4Adjusting the conductivity to 80 +/-20 mS/cm, and preparing a to-be-chromatographed liquid;
s3: and (2) carrying out hydrophobic chromatography on the liquid to be chromatographed, wherein the hydrophobic chromatography comprises the following steps:
s31: loading the liquid to be chromatographed to a hydrophobic chromatographic column;
s32: leaching for the first time: the pH of the first eluent used was 5.5 ± 0.5, said first eluent comprising: 12. + -. 2mM NaAc, 1.7. + -. 0.5mM HAc and 0.9. + -. 0.5M (NH)4)2SO4The dosage of the first leacheate is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 5 minutes;
s33: and (3) leaching for the second time: a second eluent having a pH of 5 ± 0.5 is employed, said second eluent comprising: 10. + -. 2mM NaAc, 70. + -. 5mM HAc, 0.05. + -. 0.01M arginine and 0.6. + -. 0.5M (NH)4)2SO4The dosage of the second leacheate is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 5 minutes;
s34: and (3) elution: a first eluent having a pH of 5 ± 0.5 is employed, said first eluent comprising: NaAc 10 + -2 mM, HAc 73 + -5 mM, arginine 0.05 + -0.01M, and (NH) 10 + -0.5 mM4)2SO4The dosage of the first eluent is more than or equal to 10 times of the column volume, and the retention time is more than or equal to 5 minutes; the peak receiving parameter is 250mAU/mm before peak to 50mAU/mm after peak.
In one embodiment, (NH) in step S24)2SO4Is added in the form of 1-5M aqueous solution.
In one embodiment, in step S31, the loading amount of the sample is 10-20 g/L, the temperature of the sample is 18-26 ℃, and the retention time is not less than 5 minutes.
In one embodiment, in step S31, the hydrophobic chromatography column uses a hydrophobic filler bonded with phenyl functional groups, and the particle size is 65 μm.
In one example, in step S31, the hydrophobic chromatography column is packed with TOYOPEARL Phenyl-650M.
In one embodiment, the molecular size of the nano antibody is 75 KD-85 KD.
In one embodiment, in step S1, the step of affinity chromatography comprises:
s11: loading the nano antibody harvest solution to an affinity chromatography column;
s13: leaching with a third leacheate, wherein the pH of the third leacheate is 7 +/-0.5, and the third leacheate comprises: 20 +/-2 mM Tris, 20 +/-2 mM HAc and 0.15 +/-0.05M NaCl, wherein the dosage of the third leacheate is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 5 minutes;
s13: eluting with a second eluent having a pH of 3 ± 0.5, the second eluent comprising: 20 plus or minus 2mM NaAc and 20 plus or minus 2mM HAc, wherein the dosage of the second eluent is more than or equal to 5 times of the column volume, and the retention time is more than or equal to 5 minutes; the peak receiving parameter is 50mAU/mm before peak to 50mAU/mm after peak.
In one embodiment, in step S11, the loading amount of the sample is 10-50 g/L, the temperature of the sample is 18-26 ℃, and the retention time is not less than 5 minutes.
In one embodiment, step S31 further comprises the steps of washing, sterilizing and equilibrating the column before loading it on the hydrophobic chromatography column.
In one embodiment, the step of column washing comprises: washing with injection water, wherein the dosage of the injection water is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes.
In one embodiment, the step of sterilizing comprises: and (3) washing with 0.3-1M of NaOH aqueous solution, wherein the using amount of the NaOH aqueous solution is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes.
In one embodiment, the step of balancing comprises: flushing by using a balance liquid, wherein the using amount of the balance liquid is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes; the pH of the equilibrium liquid is 5.5 +/-0.5, and the equilibrium liquid comprises: 12. + -. 2mM NaAc, 1.7. + -. 0.5mM HAc and 0.9. + -. 0.5M (NH)4)2SO4。
According to the chromatography method of the nano antibody, through reasonably setting each process step, particularly carrying out specific blending on the sample before hydrophobic chromatography, and carrying out hydrophobic chromatography on the sample by adopting specific compatible eluent and eluent, the aggregate, Host Cell Protein (HCP) and Host Cell DNA (HCD) in the sample can be effectively removed. Meanwhile, the stability of the nano antibody can be maintained in the hydrophobic chromatography process, and the formation of aggregates is reduced.
Detailed Description
The chromatographic method of the nanobody of the present invention will be described in further detail with reference to specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.
In the present invention, the numerical intervals are regarded as continuous, and include the minimum and maximum values of the range and each value between the minimum and maximum values, unless otherwise specified. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to encompass any and all subranges subsumed therein.
The percentage contents referred to in the present invention mean, unless otherwise specified, mass percentages for solid-liquid mixing and solid-solid phase mixing, and volume percentages for liquid-liquid phase mixing.
The percentage concentrations referred to in the present invention refer to the final concentrations unless otherwise specified. The final concentration refers to the ratio of the additive component in the system to which the component is added.
The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a treatment within a certain temperature range. The constant temperature process allows the temperature to fluctuate within the accuracy of the instrument control.
Unless otherwise specified, the solvents of the solutions of the present invention are all water for injection.
The abbreviations referred to in the present invention have the following meanings:
HCP: a host cell protein;
HCD (hydrogen chloride): host cell DNA;
NaAc: sodium acetate;
HAc: acetic acid;
arg: arginine;
WFI: water for injection;
ppm: concentration units, expressed as ng/mg;
ppb: concentration units, expressed in pg/mg;
m: concentration unit, which represents mol/L;
and (mM): concentration units, expressed as mmol/L;
SE-HPLC, gel filtration chromatography.
The invention provides a chromatography method of a nano antibody, which comprises the following steps:
s1: carrying out affinity chromatography on the nano antibody harvest solution, and collecting an affinity chromatography eluent;
s2: adjusting pH of the affinity chromatography eluate to 5 + -0.5 and the concentration of the nano antibody to be less than or equal to 6mg/mL, and adding (NH)4)2SO4Adjusting the conductivity to 80 +/-20 mS/cm, and preparing a to-be-chromatographed liquid;
s3: carrying out hydrophobic chromatography on the to-be-chromatographed liquid, wherein the hydrophobic chromatography comprises the following steps:
s31: loading the liquid to be chromatographed to a hydrophobic chromatographic column;
s32: leaching for the first time: the pH of the first eluent used was 5.5 ± 0.5, the first eluent comprising: 12. + -. 2mM NaAc, 1.7. + -. 0.5mM HAc and 0.9. + -. 0.5M (NH)4)2SO4The dosage of the first leacheate is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 5 minutes;
s33: and (3) leaching for the second time: the pH of the second eluent used is5 ± 0.5, the second eluate comprising: 10. + -. 2mM NaAc, 70. + -. 5mM HAc, 0.05. + -. 0.01M arginine and 0.6. + -. 0.5M (NH)4)2SO4The dosage of the second leacheate is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 5 minutes;
s34: and (3) elution: a first eluent having a pH of 5 ± 0.5 is employed, said first eluent comprising: NaAc 10 + -2 mM, HAc 73 + -5 mM, arginine 0.05 + -0.01M, and (NH) 10 + -0.5 mM4)2SO4The dosage of the first eluent is more than or equal to 10 times of the column volume, and the retention time is more than or equal to 5 minutes; the peak receiving parameter is 250mAU/mm before peak to 50mAU/mm after peak.
In one specific example, the molecular size of the nanobody is 75-85 KD. Specifically, the molecular size of nanobodies includes, but is not limited to: 75KD, 76KD, 77KD, 78KD, 79KD, 80KD, 81KD, 82KD, 83KD, 84KD, 85 KD.
Specifically, step S1 is an affinity chromatography step.
In one specific example, in step S1, the step of affinity chromatography comprises:
s11: loading the nano antibody harvest solution to an affinity chromatography column;
s13: and (3) leaching by using a third leaching solution, wherein the pH value of the third leaching solution is 7 +/-0.5, and the third leaching solution comprises: 20 plus or minus 2mM Tris, 20 plus or minus 2mM HAc and 0.15 plus or minus 0.05M NaCl, the dosage of the third eluent is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 5 minutes; further, the dosage of the third leacheate is 3-5 times of the column volume, and the retention time is 5-10 min;
s13: eluting with a second eluent having a pH of 3 + -0.5, the second eluent comprising: 20 plus or minus 2mM NaAc and 20 plus or minus 2mM HAc, the dosage of the second eluent is more than or equal to 5 times of the column volume, and the retention time is more than or equal to 5 minutes; further, the dosage of the second eluent is 5-10 times of the column volume, and the retention time is 5-10 min; the peak receiving parameter is 50mAU/mm before peak to 50mAU/mm after peak.
In one specific example, the preparation method of the nano-antibody harvest comprises the following steps: taking the cell culture solution, centrifuging, sterilizing and filtering, and collecting the supernatant.
In one specific example, in step S11, the loading amount of the sample loading is 10-50 g/L, the temperature of the sample loading is 18-26 ℃, and the retention time is more than or equal to 5 minutes. Further, the retention time is 5 min-10 min.
In one specific example, step S11 includes the steps of washing, sterilizing and equilibrating the column before loading it onto the affinity chromatography column.
In one particular example, the step of column washing comprises: the column is washed by using injection water, the using amount of the injection water is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes. Furthermore, the dosage of the water for injection is 3-5 times of the column volume, and the retention time is 3-5 min.
In one specific example, the step of sterilizing comprises: and (3) washing with 0.05-0.2M of NaOH aqueous solution, wherein the using amount of the NaOH aqueous solution is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes. Furthermore, the amount of the NaOH aqueous solution is 3-5 times of the column volume, and the retention time is 3-5 min.
In one specific example, the step of balancing comprises: flushing with a balancing solution, wherein the using amount of the balancing solution is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes; further, the using amount of the equilibrium liquid is 3-5 times of the column volume, and the retention time is 3-5 min; the pH of the equilibrium solution is 7 + -0.5, and the equilibrium solution comprises: 20. + -.2 mM Tris, 20. + -.2 mM HAc and 0.15. + -. 0.05M NaCl.
Step S2 is a sample preparation step before hydrophobic chromatography.
In one specific example, in step S2, the pH of the affinity chromatography eluent is adjusted to 5 + -0.5 by adjusting the pH of the affinity chromatography eluent to 5 + -0.5 with 1.5M to 2.5M arginine in water.
In one specific example, in step S2, the method for adjusting the concentration of nanobody in the affinity chromatography eluate to be less than or equal to 6mg/mL is to add water for injection for dilution.
In one specific example, in step S2, the conductivity is adjusted to 80. + -.20 mS/cm (NH)4)2SO4Adding the mixture in the form of 1-5M aqueous solution. Specifically, the (NH)4)2SO4The concentrations of (a) include, but are not limited to: 1M, 2M, 3M, 4M, 5M. Further, the (NH) is added slowly during the adjustment4)2SO4And continuously stirring at a low speed, wherein the speed of the low-speed stirring can be 60-120 r/min.
It will be appreciated that the sample is loaded immediately after conditioning for hydrophobic chromatography, and that each cycle of hydrophobic chromatography requires separate conditioning.
Specifically, in step S2, the pH of the affinity chromatography eluent is adjusted to 5 and the concentration is less than or equal to 6mg/mL, and then (NH) is added4)2SO4Adjusting the conductivity to 80mS/cm, and preparing the liquid to be chromatographed.
Step S3 is a hydrophobic chromatography step.
In one specific example, in step S3, the filler in the hydrophobic chromatography column is a hydrophobic filler bonded with phenyl functional groups, and the particle size range is 65 μm. Further, the filler used for hydrophobic chromatography was TOYOPEARL Phenyl-650M.
In one specific example, the hydrophobic chromatography column is subjected to column washing, sterilization and equilibration prior to loading.
In one particular example, the step of column washing comprises: the column is washed by using injection water, the using amount of the injection water is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes. Furthermore, the dosage of the water for injection is 3-5 times of the column volume, and the retention time is 3-5 min.
In one specific example, the step of sterilizing comprises: and (3) washing with 0.3-1M of NaOH aqueous solution, wherein the using amount of the NaOH aqueous solution is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes. Furthermore, the amount of the NaOH aqueous solution is 3-5 times of the column volume, and the retention time is 3-5 min. Specifically, the concentration of the aqueous NaOH solution includes, but is not limited to: 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M.
In one specific example, the step of balancing comprises: washing with a balancing solution, wherein the using amount of the balancing solution is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes; further, the using amount of the equilibrium liquid is 3-5 times of the column volume, and the retention time is 3-5 min; the pH of the equilibrium liquid is 5.5 +/-0.5, and the equilibrium liquid comprises: 12 +/-2NaAc in mM, HAc in 1.7 + -0.5 mM, and (NH) in 0.9 + -0.5M4)2SO4. Further, the pH of the equilibrium liquid was 5.5, and the equilibrium liquid contained: 12mM NaAc, 1.7mM HAc and 0.9M (NH)4)2SO4。
Step S31 is a sample application step.
In one specific example, in the step S31, the loading amount of the sample loading ranges from 10 g/L to 20g/L, the temperature of the sample loading ranges from 18 ℃ to 26 ℃, and the retention time is more than or equal to 5 minutes. Further, the retention time is 5 min-10 min.
Step S32 is a first rinsing step.
In one specific example, a first rinse solution having a pH of 5.5 is used, the first rinse solution comprising: 12mM NaAc, 1.7mM HAc and 0.9M (NH)4)2SO4The dosage of the first leacheate is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 5 minutes; further, the dosage of the first leacheate is 3-5 times of the column volume, and the retention time is 5-10 min.
Step S33 is a second rinsing step.
In one specific example, a second rinse solution having a pH of 5 is used, the second rinse solution comprising: 10mM NaAc, 70mM HAc, 0.05M Arg, and 0.6M (NH)4)2SO4The dosage of the second leacheate is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 5 minutes; further, the dosage of the second leacheate is 3-5 times of the column volume, and the retention time is 5-10 min.
Step S34 is a second rinsing step.
In one specific example, a first eluent having a pH of 5 is employed, the eluent comprising: NaAc 10mM, HAc 73mM, Arg 0.05M and (NH) 10mM4)2SO4The dosage of the first eluent is more than or equal to 10 times of the column volume, and the retention time is more than or equal to 5 minutes; further, the dosage of the first eluent is 10-30 times of the column volume, and the retention time is 5-30 min; the peak receiving parameter is 250mAU/mm before peak to 50mAU/mm after peak.
In addition, after the hydrophobic chromatography is finished, the method also comprises the steps of regenerating, disinfecting and storing the chromatographic column.
In one specific example, the step of regenerating comprises: washing with injection water, wherein the dosage of the injection water is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes; furthermore, the dosage of the water for injection is 3-5 times of the column volume, and the retention time is 3-5 min.
In one specific example, the step of sterilizing comprises: washing with 0.3-1M NaOH aqueous solution, wherein the using amount of the NaOH aqueous solution is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes; furthermore, the amount of the NaOH aqueous solution is 3-5 times of the column volume, and the retention time is 3-5 min. Specifically, the concentration of the aqueous NaOH solution includes, but is not limited to: 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M.
In one specific example, the step of saving comprises: washing with 0.05-0.25M NaOH aqueous solution, wherein the amount of the NaOH aqueous solution is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes; furthermore, the amount of the NaOH aqueous solution is 3-5 times of the column volume, and the retention time is 3-5 min. Specifically, the concentration of the aqueous NaOH solution includes, but is not limited to: 0.05M, 0.1M, 0.15M, 0.2M, 0.25M.
Specific examples are as follows.
The packing used for hydrophobic chromatography in the examples was TOYOPEARL Phenyl-650M, cat #: 0014783, respectively; the manufacturer: TOSOH.
The nanobody of the example is a type IgG4 nanobody expressed by a CHO cell line (chinese hamster ovary cells).
Example 1
This example is a chromatography method of nano-antibody, the molecular size of which is about 77 KD.
(1) The affinity chromatography steps were as follows:
1.1 column washing: washing the chromatographic column with injection water for 4 times the column volume and maintaining for 4 min;
1.2 pre-sterilization: washing the chromatographic column with 0.1M NaOH for 4 times of the column volume, and keeping the process for 4 minutes;
1.3 balance: the column was washed 4 column volumes with equilibration solution (20 mM Tris +20mM HA +0.15M NaCl, pH 7.0) and the process retention time 4 minutes;
1.4 loading: taking a cell culture solution, centrifuging, sterilizing and filtering, and taking a supernatant, wherein the loading capacity of a sample is 30g/L, the loading temperature is 20 ℃, and the retention time is 8 minutes;
1.5 leaching: eluting 20mM Tris +20mM HAc +0.15M NaCl with an equilibrium solution, pH 7.0) to wash the chromatographic column by at least 4 times of the column volume, wherein the process retention time is not less than 8 minutes;
1.6 elution: the column was washed 8 column volumes with eluent (20 mM NaAc +20mM HAc, pH 3.0) for a process retention time of 8 minutes; the peak collecting parameter of the eluent is 50mAU/mm before the peak and 50mAU/mm after the peak (the model of detection equipment: AKTA Avant150, supplier Cytiva); obtaining an affinity chromatography eluent;
1.7 regeneration: washing the chromatographic column with 0.5M acetic acid solution for 4 times the column volume, and maintaining for 4 min;
1.8, disinfection: washing the chromatographic column with 0.1M NaOH for 4 times of the column volume, and keeping the process for 4 minutes;
1.9 preservation: the column was flushed 4 column volumes with 2% benzyl alcohol and the process retention time was 4 minutes.
(2) The hydrophobic chromatography steps were as follows:
2.1 column washing: washing the chromatographic column with injection water for 4 times the column volume and maintaining for 4 min;
2.2 pre-sterilization: washing the chromatographic column with 0.5M NaOH for 4 times of the column volume, and keeping the process for 43 minutes;
2.3 balance: equilibration solution (12 mM NaAc +1.7mM HAc +0.9M (NH) was used4)2SO4pH5.5) washing the chromatographic column for 4 times of the column volume, and the process retention time is 4 minutes;
2.4 loading: taking the sample with the pH value of the affinity chromatography eluent adjusted back to 5.0, adding WFI to dilute the sample until the concentration of the nano antibody is 5mg/mL, and slowly adding 3M (NH) while continuously stirring at a low speed (80 revolutions per minute)4)2SO4The conductivity was adjusted to 80mS/cm and the sample was immediately applied after the adjustment. The loading capacity of the sample is 15g/L, the loading temperature is 20 ℃, and the retention time is 8 minutes;
2.5 rinsing 1: spraying with the balance liquidWash 1 (12 mM NaAc +1.7mM HAc +0.9M (NH)4)2SO4pH5.5) washing the chromatographic column for 4 times of the column volume, and the process retention time is 8 minutes;
2.6 rinsing 2: elution 2 (10 mM NaAc +70Mm HAc +0.05M Arg +0.6M (NH) using eluent 24)2SO4pH5.0) washing the chromatographic column for 4 times of the column volume, and the process retention time is 8 minutes;
2.7 elution: elution was performed with eluent (10 mM NaAc +73mM HAc +0.05M Arg +10mM (NH)4)2SO4pH5.0) washing the chromatographic column for 15 times of the column volume, and the process retention time is 10 minutes; the peak collecting parameter of the eluent is 250mAU/mm before the peak and 50mAU/mm after the peak (the model of detection equipment: AKTA Avant150, supplier Cytiva); obtaining a hydrophobic chromatography eluent;
2.8 regeneration: washing the chromatographic column with WFI for 4 times the column volume and 4 min process retention time;
2.9, disinfection: washing the chromatographic column with 0.5M NaOH for 4 times of the column volume, and keeping the process for 4 minutes;
2.10 preservation: the column was flushed with 0.1M NaOH for 4 column volumes and the process retention time was 4 minutes.
The quality tests of the affinity chromatography eluent and the hydrophobic chromatography eluent of the example were carried out separately, and the results are shown in the following table 1:
TABLE 1
Note: the mass requirement ranges are HCP <100ppm and HCD <10 ppb.
As can be seen from Table 1, the HCP and HCD residues can be significantly reduced by performing the crude purification by hydrophobic chromatography, and are respectively 41ppm and 6ppm, the purity of the SE-HPLC main peak can reach more than 99%, and the aggregate content is only 0.5%.
Comparative example 1
The comparative example is a chromatography method of a nano antibody, and the steps are the same as those of example 1, and the main differences are that: in each step of step (2), sodium chloride is used instead of ammonium sulfate.
The quality of the affinity chromatography eluate (same as example 1) and the hydrophobic chromatography eluate of the comparative example were measured, respectively, and the results are shown in the following table 2:
TABLE 2
Note: the mass requirement ranges are HCP <100ppm and HCD <10 ppb.
As can be seen from table 2, comparative example 1 failed to effectively remove HCP and HCD, while the aggregates increased.
Comparative example 2
The comparative example is a chromatography method of a nano antibody, and the steps are the same as those of example 1, and the main differences are that: in the step (2), a phosphate buffer system is adopted, and sodium chloride is used for replacing ammonium sulfate and arginine.
Specifically, (2) the hydrophobic chromatography step is as follows:
2.1 column washing: washing the chromatographic column with injection water for 4 times the column volume and maintaining for 4 min;
2.2 pre-sterilization: washing the chromatographic column with 0.5M NaOH for 4 times of the column volume, and keeping the process for 43 minutes;
2.3 balance: the column was flushed 4 column volumes with equilibration solution (20 mM PB +0.9M NaCl, pH 7.0) for a process retention time of 4 minutes;
2.4 loading: taking the sample with the pH value of the affinity chromatography eluent adjusted back to 5.0, adding WFI to dilute the sample until the concentration of the nano antibody is 5mg/mL, and slowly adding 3M (NH) while continuously stirring at a low speed (80 revolutions per minute)4)2SO4The conductivity was adjusted to 80mS/cm and the sample was immediately applied after the adjustment. The loading capacity of the sample is 15g/L, the loading temperature is 20 ℃, and the retention time is 8 minutes;
2.5 rinsing 1: eluting 1 (20 mM PB +0.9M NaCl, pH7.0) by using the equilibrium solution to wash the chromatographic column by 4 times of the column volume, and keeping the process for 8 minutes;
2.6 rinsing 2: elution 2 (20 mM PB +0.5M NaCl, pH 7.0) was performed with eluent 2 to wash the column for 4 column volumes with a process retention time of 8 minutes;
2.7 elution: elution with eluent (20 mM PB +0.1M NaCl, pH 7.0) washes the column for 15 column volumes with a process retention time of 10 minutes; the peak collecting parameter of the eluent is 250mAU/mm before the peak and 50mAU/mm after the peak (the model of detection equipment: AKTA Avant150, supplier Cytiva); obtaining a hydrophobic chromatography eluent;
2.8 regeneration: washing the chromatographic column with WFI for 4 times the column volume and 4 min process retention time;
2.9, disinfection: washing the chromatographic column with 0.5M NaOH for 4 times of the column volume, and keeping the process for 4 minutes;
2.10 preservation: the column was flushed with 0.1M NaOH for 4 column volumes and the process retention time was 4 minutes.
The quality of the affinity chromatography eluate (same as example 1) and the hydrophobic chromatography eluate of the comparative example were measured, respectively, and the results are shown in the following table 3:
TABLE 3
Note: the mass requirement ranges are HCP <100ppm and HCD <10 ppb.
As can be seen from table 3, comparative example 2 failed to effectively remove HCP and HCD, while the aggregates increased.
Comparative example 3
The comparative example is a chromatography method of a nano antibody, and the steps are the same as those of example 1, and the main differences are that: in step 2.4, the sample with the pH value of the affinity chromatography eluent adjusted back to 7.0 is taken.
The quality of the affinity chromatography eluate (same as example 1) and the hydrophobic chromatography eluate of the comparative example were measured, respectively, and the results are shown in the following table 4:
TABLE 4
Note: the mass requirement ranges are HCP <100ppm and HCD <10 ppb.
As can be seen from table 4, comparative example 3 failed to effectively remove HCP and HCD, while the aggregates increased.
Comparative example 4
The comparative example is a chromatography method of a nano antibody, and the steps are the same as those of example 1, and the main differences are that: in step 2.4, 3M (NH) is added4)2SO4Adjusted to a conductance of 30 mS/cm.
The quality of the affinity chromatography eluate (same as example 1) and the hydrophobic chromatography eluate of the comparative example were measured, respectively, and the results are shown in the following table 5:
TABLE 5
Note: the mass requirement ranges are HCP <100ppm and HCD <10 ppb.
As can be seen from table 5, comparative example 4 failed to effectively remove HCP and HCD, while the aggregates increased.
Comparative example 5
The comparative example is a chromatography method of a nano antibody, and the steps are the same as those of example 1, and the main differences are that: in each step of step (2), Arg is not added.
The quality of the affinity chromatography eluate (same as example 1) and the hydrophobic chromatography eluate of the comparative example were measured, respectively, and the results are shown in the following table 6:
TABLE 6
Note: the mass requirement ranges are HCP <100ppm and HCD <10 ppb.
As can be seen from table 6, comparative example 5 failed to effectively remove HCP and HCD, while the aggregates increased.
Comparative example 6
The comparative example is a chromatography method of a nano antibody, and the steps are the same as those of example 1, and the main differences are that: in each step of step (2), the concentration of Arg was increased to 0.1M.
The quality of the affinity chromatography eluate (same as example 1) and the hydrophobic chromatography eluate of the comparative example were measured, respectively, and the results are shown in the following table 7:
TABLE 7
Note: the mass requirement ranges are HCP <100ppm and HCD <10 ppb.
As can be seen from table 7, comparative example 6 failed to effectively remove HCP and HCD, while the aggregates increased.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, so as to understand the technical solutions of the present invention specifically and in detail, but not to be understood as the limitation of the protection scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments, are within the scope of the appended claims. Therefore, the protection scope of the patent of the invention is subject to the content of the appended claims, and the description can be used for explaining the content of the claims.
Claims (10)
1. A chromatography method of a nano antibody is characterized by comprising the following steps:
s1: performing affinity chromatography on a nano antibody harvest solution, and collecting an affinity chromatography eluent, wherein the nano antibody harvest solution contains IgG4 type nano antibodies expressed by a CHO cell line;
s2: adjusting the pH of the affinity chromatography eluent to 5 +/-0.5 and the concentration of the nano antibody to be less than or equal to 6mg/mL, and then adding (NH)4)2SO4Adjusting the conductivity to 80 +/-20 mS/cm, and preparing a to-be-chromatographed liquid;
s3: and (2) carrying out hydrophobic chromatography on the liquid to be chromatographed, wherein the hydrophobic chromatography comprises the following steps:
s31: loading the to-be-chromatographed liquid to a hydrophobic chromatographic column, wherein the hydrophobic chromatographic column adopts TOYOPEARL Phenyl-650M as a filler;
s32: leaching for the first time: the pH of the first eluent used was 5.5 ± 0.5, said first eluent comprising: 12. + -. 2mM NaAc, 1.7. + -. 0.5mM HAc and 0.9. + -. 0.5M (NH)4)2SO4The dosage of the first leacheate is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 5 minutes;
s33: and (3) leaching for the second time: a second eluent having a pH of 5 ± 0.5 is employed, said second eluent comprising: 10. + -. 2mM NaAc, 70. + -. 5mM HAc, 0.05. + -. 0.01M arginine and 0.6. + -. 0.5M (NH)4)2SO4The dosage of the second leacheate is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 5 minutes;
s34: and (3) elution: a first eluent having a pH of 5 ± 0.5 is employed, said first eluent comprising: NaAc 10 + -2 mM, HAc 73 + -5 mM, arginine 0.05 + -0.01M, and (NH) 10 + -0.5 mM4)2SO4The dosage of the first eluent is more than or equal to 10 times of the column volume, and the retention time is more than or equal to 5 minutes; the peak receiving parameter is 250mAU/mm before peak to 50mAU/mm after peak.
2. The method for chromatography of nanobodies according to claim 1, wherein the step S2 is (NH)4)2SO4Is added in the form of 1-5M aqueous solution.
3. The chromatography method of nanobodies according to claim 1, wherein in step S31, the loading amount of the sample ranges from 10 to 20g/L, the temperature of the sample is from 18 ℃ to 26 ℃, and the retention time is not less than 5 minutes.
4. The chromatography method of the nanobody of claim 1, wherein the molecular size of the nanobody is 75-85 KD.
5. The method for chromatography of nanobodies according to any one of claims 1 to 4, wherein in step S1, the step of affinity chromatography comprises:
s11: loading the nano antibody harvest solution to an affinity chromatography column;
s13: leaching with a third leacheate, wherein the pH of the third leacheate is 7 +/-0.5, and the third leacheate comprises: 20 +/-2 mM Tris, 20 +/-2 mM HAc and 0.15 +/-0.05M NaCl, wherein the dosage of the third leacheate is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 5 minutes;
s13: eluting with a second eluent having a pH of 3 ± 0.5, the second eluent comprising: 20 plus or minus 2mM NaAc and 20 plus or minus 2mM HAc, wherein the dosage of the second eluent is more than or equal to 5 times of the column volume, and the retention time is more than or equal to 5 minutes; the peak receiving parameter is 50mAU/mm before peak to 50mAU/mm after peak.
6. The method for chromatography of nanobodies according to claim 5, wherein in step S11, the loading amount of the sample ranges from 10 to 50g/L, the temperature of the sample is from 18 ℃ to 26 ℃, and the retention time is not less than 5 minutes.
7. The method for chromatography of nanobodies according to any one of claims 1 to 4, wherein the step of washing, sterilizing and equilibrating the column is further included before loading the hydrophobic chromatography column in step S31.
8. The nanobody chromatography method according to claim 7, wherein the column washing step comprises: washing with injection water, wherein the dosage of the injection water is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes.
9. The nanobody chromatography method of claim 7, wherein the step of sterilizing comprises: and (3) washing with 0.3-1M of NaOH aqueous solution, wherein the using amount of the NaOH aqueous solution is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes.
10. The nanobody chromatography method of claim 7, wherein the step of equilibrating comprises: flushing by using a balance liquid, wherein the using amount of the balance liquid is more than or equal to 3 times of the column volume, and the retention time is more than or equal to 3 minutes; the pH of the equilibrium liquid is 5.5 +/-0.5, and the equilibrium liquid comprises: 12. + -. 2mM NaAc, 1.7. + -. 0.5mM HAc and 0.9. + -. 0.5M (NH)4)2SO4。
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